Drug therapies for many cancers continue to be inadequate, having either limited efficacy, prohibitive toxicities, or in many cases both. As an example, effective therapies are sorely needed for non-small cell lung cancers (NSCLC), of which there are over 162,000 deaths per year according to the National Cancer Institute. Eighty percent of the over 200,000 new diagnoses of lung cancer each year are non-small cell carcinomas. While some patients are successful candidates for surgical resection or radiation therapy, most patients have disseminated disease at the time of diagnosis and are therefore not candidates for these approaches. Most patients diagnosed in the later stages will need to be treated with a variety of therapies including chemotherapies and biologically targeted therapies, neither of which work well for the majority of patients. Results of standard treatment are poor except for the most localized cancers, and currently, no single chemotherapy or biologic regimen can be recommended for routine use. Furthermore, according to the National Cancer Institute, there are nearly 12,000 new diagnoses of myeloid leukemia and over 9,000 deaths from this cancer each year. Thirty to 40% of patients will not attain complete remission of this disease following standard chemotherapy, and only 25% of those attaining complete remission are expected to live longer than 3 years. Thus as with most cancers, there continues to be a need for new therapies that can keep the cancer in remission and increase survival.
There are several new, biologically targeted agents under investigation for NSCLC and other cancers in the hopes that these new agents will expand the pool of patients who respond to and receive a survival benefit from these therapies. In recent years, inhibition of specific cancer-associated tyrosine kinases has emerged as an important approach for cancer therapy. Tyrosine kinases as mediators of cell signaling, play a role in many diverse physiological pathways including cell growth and differentiation. Deregulation of tyrosine kinase activity can result in cellular transformation leading to the development of human cancer. Of the nearly thirty novel cancer targets extensively studied in the past ten years, one third of these are tyrosine or other kinases. Of the ten truly novel anti-cancer therapies approved in the past five years, five of these have been directed against receptor tyrosine kinases (RTKs). In fact, many cancer treatment protocols now use a combination of traditional chemotherapy drugs and novel biologically targeted agents, several of which inhibit tyrosine kinase activity or downstream signaling pathways. For example, a small molecule drug that inhibits the abl tyrosine kinase has led to significant improvement in outcomes for patients with chronic myelogenous leukemia Inhibitors of other tyrosine kinases, including the Flt-3, EGFR, and PDGF receptor tyrosine kinases are also in clinical trials.
The Axl receptor tyrosine kinase (Axl), originally identified as a protein encoded by a transforming gene from primary human myeloid leukemia cells, is overexpressed in a number of different tumor cell types and transforms NIH3T3 fibroblasts (O'Bryan et al., Mol. Cell Bio. 11:5016-5031 (1991)). Axl signaling has been shown to favor tumor growth through activation of proliferative and anti-apoptotic signaling pathways, as well as through promotion of angiogenesis and tumor invasiveness. Axl is associated with the development and maintenance of various cancers including lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, gliomas, melanoma, prostate cancer, breast cancer, gastric cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer, among others. Furthermore, in some cancer types, particularly non-small cell lung cancer (NSCLC), myeloid leukemia, and gastric cancers, the over-expression of this cell signaling molecule indicates a poor prognosis for the patient. Researchers have found that siRNA knockdown of Axl in NSCLC cell lines reduced invasive capacity of the tumor cells (Holland et al., 2005, Cancer Res. 65:9294-9303). Vajkoczy et al. have shown that expression of a dominant-negative Axl construct decreased brain tumor proliferation and invasion (Vajkoczy et al., 2006, PNAS 15:5799-804; European Patent Publication No. EP 1 382 969 A1). Furthermore, in clinical patient samples of NSCLC, Axl protein over-expression has been statistically associated with lymph node involvement and advanced clinical stage of disease.
Axl signaling also plays important roles in spermatogenesis (Lu et al., 1999, Nature 398:723-728), immunity (Lu and Lemke, 2001, Science 293: 306-311; Scott et al, 2001, Nature 411: 207-211), platelet function (Angelillo-Scherrer et al, 2001, 2005) and even kidney pathology (Yanagita et al, 2002, J Clin Invest 110:239-246).
Axl is related to two other receptor tyrosine kinases, Mer and Tyro-3. Axl, Mer, and Tyro-3 are all expressed in a spectrum of hematopoeitic, epithelial, and mesenchymal cell lines. Each protein has been shown to have the capability to transform cells in vitro. Axl, Mer, and Tyro-3 are all activated by the ligand Gas6. Gas6 is structurally similar to Protein S, a cofactor for anticoagulant Protein C, and shares 48% protein identity with Protein S, which has also been shown to be a binding ligand of at least Mer and Tyro-3. Gas6 plays a role in coagulation (Angelillo-Scherrer et al., Nature Medicine 7:215-21 (2002)), and Gas6 antibodies may be used to protect wild type mice against fatal thromboembolism (Angelillo-Scherrer et al., (2002)). Mice with an inactivated Gas6 gene (i.e., Gas6 knockout) have platelet dysfunction that prevents venous and arterial thrombosis. These knockout mice are protected against (have decreased mortality against) fatal collagen/epinephrine induced thromboembolism and inhibited ferric chloride-induced thrombosis in vivo. Gas6 amplifies platelet aggregation and secretion response of platelets to known agonists (Chen et al., Aterioscler. Thromb. Vasc. Biol. 24:1118-1123 (2004)). The platelet dysfunction caused by Gas6 is thought to be mediated through the Axl, Mer, or Tyro-3. In addition, mice with an inactivated Mer gene, inactivated Axl gene, or an inactivated Tyro-3 gene, all have platelet dysfunction, as well as decreased mortality against thromboembolism (by both statis-induced thrombosis in the inferior vena cava and by collagen-epinephrine induced pulmonary embolism (Angelillo-Scherrer et al., 2005, J. Clin Invest. 115:237-246). Therefore, in addition to its association with neoplastic disease, Axl is also involved in blood clotting.
Various types of thrombosis and the complications associated with thrombosis represent a major cause of morbidity and death in the world. Although there are a variety of agents to thin the blood, all have the potential for bleeding complications, and with the exception of heparin (which itself cannot be tolerated by many patients), are largely irreversible. Malignant cellular growth or tumors (cancer) are also a leading cause of death worldwide. Accordingly, the development of effective therapy for cardiovascular and neoplastic disease is the subject of a large body of research. Although a variety of innovative approaches to treat and prevent such diseases have been proposed, these diseases continue to have a high rate of mortality and may be difficult to treat or relatively unresponsive to conventional therapies. Therefore, there is a continued need in the art for new therapies that can effectively target and prevent or treat these diseases. Because it is generally the case in cancer therapy that no single agent can successfully treat a patient, new agents can continue to be developed and ultimately used in combination with other agents to affect the best outcome for patients.